Abstract

Purified human liver carboxylesterase (hCE-1) catalyzes the hydrolysis of cocaine to form benzoylecgonine, the deacetylation of heroin to form 6-acetylmorphine, and the ethanol-dependent transesterification of cocaine to form cocaethylene. In this study, the binding affinities of cocaine, cocaine metabolites and analogs, heroin, morphine, and 6-acetylmorphine for hCE-1 were evaluated by measuring their kinetic inhibition constants with 4-methylumbelliferyl acetate in a rapid spectrophotometric assay. The naturally occurring (R)-(−)-cocaine isomer displayed the highest affinity of all cocaine and heroin analogs or metabolites. The pseudo- or allopseudococaine isomers of cocaine exhibited lower affinity indicating that binding to the enzyme is stereoselective. The methyl ester, benzoyl, and N-methyl groups of cocaine play important roles in binding because removal of these groups increasedK_i values substantially. Compounds containing a variety of hydrophobic substitutions at the benzoyl group of cocaine bound to the enzyme with high affinity. The highK_i value obtained for cocaethylene relative to cocaine is consistent with weaker binding to the esterase and a longer elimination half-life reported for the metabolite. The spectrophotometric competitive inhibition assay used here represents an effective method to identify drug or environmental esters metabolized by carboxylesterases like hCE-1.